This invention relates to the field of antimicrobial agents and to methods for identification and characterization of potential antimicrobial agents. More particularly, this invention relates to antimicrobial agents for which the mode of action involves cellular efflux pumps and the regulation of efflux pumps.
The following background material is not admitted to be prior art to the pending claims, but is provided only to aid the understanding of the reader.
Antibiotics have been effective tools in the treatment of infectious diseases during the last half century. From the development of antibiotic therapy to the late 1980s there was almost complete control over bacterial infections in developed countries. The emergence of resistant bacteria, especially during the late 1980s and early 1990s, is changing this situation. The increase in antibiotic resistant strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs. (B. Murray, 1994, New Engl. J. Med. 330:1229-1230.)
The constant use of antibiotics in the hospital environment has selected bacterial populations that are resistant to many antibiotics. These populations include opportunistic pathogens that may not be strongly virulent but that are intrinsically resistant to a number of antibiotics. Such bacteria often infect debilitated or immunocompromised patients. The emerging resistant populations also include strains of bacterial species that are well known pathogens, which previously were susceptible to antibiotics. The newly acquired resistance is generally due to DNA mutations, or to resistance plasmids (R plasmids) or resistance-conferring transposons transferred from another organism. Infections by either type of bacterial population, naturally resistant opportunistic pathogens or antibiotic-resistant pathogenic bacteria, are difficult to treat with current antibiotics. New antibiotic molecules which can override the mechanisms of resistance are needed.
Bacteria have developed several different mechanisms to overcome the action of antibiotics. These mechanisms of resistance can be specific for a molecule or a family of antibiotics, or can be non-specific and be involved in resistance to unrelated antibiotics. Several mechanisms of resistance can exist in a single bacterial strain, and those mechanisms may act independently or they may act synergistically to overcome the action of an antibiotic or a combination of antibiotics. Specific mechanisms include degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug target (B. G. Spratt, Science 264:388 (1994)). There are, however, more general mechanisms of drug resistance, in which access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the cell or by increasing the efflux of the drug from the cell to the outside medium. Both mechanisms can lower the concentration of drug at the target site and allow bacterial survival in the presence of one or more antibiotics which would otherwise inhibit or kill the bacterial cells. Some bacteria utilize both mechanisms, combining a low permeability of the cell wall (including membranes) with an active efflux of antibiotics. (H. Nikaido, Science 264:382-388 (1994)).
In some cases, antibiotic resistance due to low permeability is related to the structure of the bacterial membranes. In general, bacteria can be divided into two major groups based on the structure of the membranes surrounding the cytoplasm. Gram-positive (G+) bacteria have one membrane, a cytoplasmic membrane. In contrast, Gram-negative (Gxe2x88x92) bacteria have two membranes, a cytoplasmic membrane and an outer membrane. These bacterial membranes are lipid bilayers which contain proteins and may be associated with other molecules. The permeability of bacterial membranes affects susceptibility/resistance to antibiotics because, while there are a few molecular targets of antibiotics, e.g., penicillin-binding proteins, that are accessible from the outer leaflet of the cytoplasmic membranes, the principal targets for antibiotics are in the cytoplasm or in the inner leaflet of the cytoplasmic membrane. Therefore for an antibiotic which has a target in the cytoplasmic membrane, in Gram-negative bacteria that antibiotic will first need to cross the outer membrane. For a target in the cytoplasm, an antibiotic will need to cross the cytoplasmic membrane in Gram-positive bacteria, and both the outer and cytoplasmic membranes in Gram-negative bacteria. For both membranes, an antibiotic may diffuse through the membrane, or may cross using a membrane transport system.
For Gram-negative bacteria, the lipid composition of the outer membrane constitutes a significant permeability barrier. The outer layer of this outer membrane contains a lipid, lipopolysaccharide (LPS), which is only found in the outer membrane of Gram-negative bacteria The lipid layer of the outer membrane is highly organized in a quasi-crystalline fashion and has a very low fluidity. Because of the low fluidity of the lipid layer of the outer membrane, even lipophilic antibiotics will not diffuse rapidly through the lipid layer. This has been shown experimentally, hydrophobic probe molecules have been shown to partition poorly into the hydrophobic portion of LPS and to permeate across the outer membrane bilayer at about one-fiftieth to one-hundredth the rate through the usual phospholipid bilayers (like the cytoplasmic membrane bilayer).
Some antibiotics may permeate through water-filled porin channels or through specific transport systems. Many of the porin channels, however, provide only narrow diameter channels which do not allow efficient difflusion of the larger antibiotic molecules. In addition, many porin channels are highly hydrophilic environments, and so do not efficiently allow the passage of hydrophobic molecules. Thus, the outer membrane acts as a molecular sieve for small molecules. This explains, in part, why Gram-negative bacteria are generally less susceptible to antibiotics than Gram-positive bacteria, and why Grain-negative bacteria are generally more resistant to large antibiotics, such as glycopeptides, that cannot cross the outer membrane.
The cytoplasmic membrane also provides a diffusion barrier for some antibiotics. However, since the fluidity of the lipid layer of the cytoplasmic membrane is higher than that of the outer membrane of Gram-negative bacteria, drugs that show some lipophilicity will be able to permeate through the lipid layer. Other drugs, such as phosphonomycin or D-cycloserine that have very low solubility in a lipophilic environment will cross the cytoplasmic membrane by using a transport system. In this case, though, if the transport system is not synthesized, the bacteria will become resistant to the drug (Peitz et al., 1967, Biochem. J. 6: 2561).
Decreasing the permeability of the outer membrane, by reducing either the number of porins or by reducing the number of a certain porin species, can decrease the susceptibility of a strain to a wide range of antibiotics due to the decreased rate of entry of the antibiotics into the cells. However, for most antibiotics, the half-equilibration times are sufficiently short that the antibiotic could exert its effect unless another mechanism is present. Efflux pumps are an example of such other mechanism. Once in the cytoplasm or periplasm a drug can be transported back to the outer medium. This transport is mediated by efflux pumps, which are constituted of proteins. Different pumps can efflux specifically a drug or group of drugs, such as the NorA system that transports quinolones, or Tet A that transports tetracyclines, or they can efflux a large variety of molecules, such as certain efflux pumps of Pseudomonas aeruginosa. In general, efflux pumps have a cytoplasmic component and energy is required to transport molecules out of the cell. Some efflux pumps have a second cytoplasmic membrane protein that extends into the periplasm. At least some efflux pumps of P. aeruginosa have a third protein located in the outer membrane.
Efflux pumps are involved in antibiotic resistance since, in some cases, they can remove a significant fraction of the antibiotic molecules which manage to enter the cells, thereby maintaining a very low intracellular antibiotic concentration. To illustrate, P. aeruginosa laboratory-derived mutant strain 799/61, which does not produce any measurable amounts of efflux pump is 8 to 10 fold more susceptible to tetracycline and ciprofloxacin than the parent strain P. aeruginosa 799, which synthesizes efflux pumps. Also, null mutants of mexA, the cytoplasmic component of a P. aeruginosa efflux pump, are more susceptible to antibiotics than the wild type.
The physiological role of efflux pumps has not been clearly defined yet. They are involved in drug resistance but they also are involved in the normal physiology of the bacterial cell. The efflux pump coded in the mexA operon of P. aeruginosa has been shown to be regulated by the iron content of the medium, and it is co-regulated with the synthesis of the receptors of siderophores. Siderophores are molecules that are needed for bacterial growth under iron starvation conditions, such as during infection of an animal. They are synthesized in the cytoplasm and exported when the bacterial cell needs iron. Siderophores scavenge iron within the infected animal and return the iron to the microbe to be used for essential microbial processes. Since there is essentially no free iron in the bodies of animals, including the human body, the production of siderophores by infecting bacteria is an important virulence factor for the progress of the infection.
Even organisms normally surrounded by a cell envelope of relatively high permeability can develop resistance by decreasing the permeability of the envelope. When an agent mainly diffises across the barrier through a specific channel, mutational loss of the channel can be an efficient mechanism for resistance. A xe2x80x9cnonclassicalxe2x80x9d beta-lactam compound, imipenem, shows an exceptional activity against P. aeruginosa, mainly because this agent diffuses though a specific channel, OprD, whose physiological function appears to be that of the transport of basic amino acids. However, P. aeruginosa could become resistant to imipenem by simply losing the oprD channel, and currently a large fraction of P. aeruginosa strains isolated from the hospital environment are resistant as a result of this modification. In a similar manner, beta-lactam compounds designed to mimic iron-chelating compounds (siderophores) during their transport through the outer membranes are known to select mutants that are defective in the specific transport of these siderophores.
In summary, the above discussion indicates that cellular factors affecting transport (both active and passive transport) of antibiotics into bacterial cells are important components of antibiotic resistance for many bacterial species.
This invention concerns particular compounds which are efflux pump inhibitors, and which are therefore compounds which inhibit cellular efflux pumps of bacteria or other microbes. Such efflux pumps export substrate molecules from the cytoplasm in an energy-dependent manner, and the exported substrate molecules can include antibacterial agents. Such efflux pump inhibitors are useful, for example, for treating microbial infections by reducing the export of a co-administered antimicrobial agent or by preventing the export of a compound synthesized by microbes (e.g, bacteria) to allow or improve their growth. An example of reducing the export of such a compound is inhibiting iron availability for the microbe by reducing the export of siderophores. Thus, this invention also provides compositions which include such efflux pump inhibitors and methods for treating microbial infections using those compositions.
The general identification and use of efflux pump inhibitors is described in co-pending U.S. patent applications, Trias et al., EFFLUX PUMP INHIBITORS, application Ser. No. 08/427,088, filed Apr. 21, 1995; Trias et al., EFFLUX PUMP INHIBITORS, Appl. No. not yet assigned, filed Jul. 22, 1997; and Trias et al., PCT Application PCT/US96/05469, International Publication WO96/33285, which are hereby incorporated by reference in their entireties including drawings. Screening methods described therein were used to identity some of the efflux inhibitor compounds of the present invention, and additional compounds were synthesized and tested which were structurally related to the active compounds identified through screening.
The efflux pump inhibitors of the present invention have structures which are shown by the generic structure in Table 1 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration. Even racemic materials fulfill the structural generics descriptions.
In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally given as a range. For example, an alkyl group containing from 1 to 4 carbon atoms is indicated as alkyl (C1-C4), or as (C1-4) alkyl. Such a range reference is intended to include specific references to groups having each of the number of atoms within the specified range including the endpoints. Other numbers of atoms and other types of atoms are indicated in a similar manner. For example, C1-4 includes each of C1, C2, C3, and C4 individually and any subgroup of the range.
Unless otherwise indicated, the term xe2x80x9calkylxe2x80x9d refers to a branched or unbranched aliphatic hydrocarbon group, preferably having from 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Preferably the hydrocarbon group is saturated. The alkyl group may optionally be substituted, and some preferred substituents include alkoxy, alkylthio, halogen, amino, monosubstituted amino, disubstituted amino, and carboxy groups.
The term xe2x80x9clower alkylxe2x80x9d refers to an aliphatic hydrocarbon having 1 to 6 carbons, and preferably 1 to 4 carbon atoms. The lower alkyl group may optionally be substituted; preferred substituents include alkoxy, alkylthio, halogen, amino, monosubstituted amino, disubstituted amino, and carboxy.
The term xe2x80x9cbranched alkylxe2x80x9d refers to a branched aliphatic hydrocarbon. The branched alkyl group is preferably 3 to 10 carbons, and most preferably 3 to 6 carbons. The branched alkyl group may optionally be substituted and some preferred substituents include alkoxy, alkylthio, halogen, amino, monosubstituted amino, disubstituted amino, and carboxy.
The term xe2x80x9chaloalkylxe2x80x9d refers to a lower alkyl group which is substituted with a halogen. Thus, the term xe2x80x9cfluoroalkylxe2x80x9d refers to a lower alkyl group which is substituted with a fluorine. The term xe2x80x9cperfluoroalkylxe2x80x9d refers to a lower alkyl group which is substituted with a fluorine atom in every available position except for where the lower alkyl group is attached to the main chain.
The term xe2x80x9ccarboxyalkylxe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94COOH, where R is an alkyl moiety, preferably a saturated alkyl, and where n is 0-5.
The term xe2x80x9chydroxyalkylxe2x80x9d refers to a chemical moiety with the formula xe2x80x94(R)nxe2x80x94OH, where R is an alkyl moiety and where n is 1-4.
The term xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkyloxyxe2x80x9d refers to a chemical substituent of formula xe2x80x94OR, where R is a saturated or unsaturated lower alkyl moiety.
The term xe2x80x9calkylthioxe2x80x9d refers to a chemical substituent of formula xe2x80x94SR, where R is hydrogen or a saturated or unsaturated lower alkyl moiety.
The term xe2x80x9carylxe2x80x9d refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl groups (e.g. pyridine). The aryl group is preferably 6 to 14 carbons, more preferably 6 to 10 carbons. Aryl moieties include monocyclic, bicyclic, and tricyclic rings, where each ring has preferably five or six members. The aryl moiety may be optionally monosubstituted or disubstituted independently with lower alkyl or alkenyl, hydroxyl, alkoxy, alkylthio, halogen, haloalkyl, mercapto, amino, monosubstituted amino, and disubstituted amino.
The term xe2x80x9ccarbocyclicxe2x80x9d refers to a compound or group which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one non-carbon atom. A xe2x80x9ccycloalkanexe2x80x9d or xe2x80x9ccyclic alkanexe2x80x9d or xe2x80x9ccycloalkylxe2x80x9d is a carbocyclic group in which the ring is an optionally substituted cyclic aliphatic hydrocarbon, i.e., a cyclic alkyl group preferably with 3, 4, 5, or 6 ring carbons. Thus, a xe2x80x9ccyclopropylxe2x80x9d group has 3 ring carbon atoms.
A xe2x80x9cheterocycloalkanexe2x80x9d or xe2x80x9cheterocycloalkylxe2x80x9d refers to a heterocyclic group in which the carbon atom portion of the ring or rings is optionally substituted alkyl, preferably lower alkyl.
Thus, the term xe2x80x9cazaheterocyclexe2x80x9d refers to a heterocyclic group which includes at least one nitrogen atom in a ring. Preferably the azaheterocyclic group is a N-morpholinyl, N-piperazinyl, N-pyrrolidinyl, N-imidazolyl, N-pyrrolyl, N-pyrazolyl, N-triazolyl, and N-tetrazolyl group. The azaheterocyclic group may also be substituted as recognized in the art, forming a substituted azaheterocycle, preferably a 2-(or 3-) lower alkylmorpholinyl, 2-(3- or 4-) lower alkylpiperazinyl, 2-(or 3-) lower alkylpyrrolidinyl, 2-(or 3-) lower alkylmorpholinyl, 2-(or 3-) lower alkylpyrrolyl group.
xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to F, Br, Cl, or I, but is preferably F or Br, and more preferably is F.
xe2x80x9cHydroxylxe2x80x9d or xe2x80x9chydroxyxe2x80x9d refers to the group xe2x80x94OH.
The term xe2x80x9caminoxe2x80x9d means the group NRRxe2x80x2, where R and Rxe2x80x2 may independently be alkyl or hydrogen or hydroxyl, but preferably are hydrogen. The term xe2x80x9cmonosubstituted aminoxe2x80x9d refers to an amino group in which one of R or Rxe2x80x2 is alkyl. The term xe2x80x9cdisubstituted aminoxe2x80x9d refers to an amino group in which R and Rxe2x80x2 are each independently alkyl or hydroxyl.
The term xe2x80x9cthienylxe2x80x9d refers to a group which has the core ring structure of Structure A below. The thienyl group may be attached to the rest of the molecule through position 2 or 3 on the ring and may be optionally independently substituted with one or more lower alkyl or alkenyl, hydroxy, alkoxy, alkylthio, mercapto, halogen, haloalkyl, amino, monosubstituted amino, or disubstituted amino (substituents as indicated for group R3 in Table 1). 
The term xe2x80x9cfurylxe2x80x9d refers to a group which has the core ring structure of Structure B. The furyl group may be attached to the rest of the molecule through position 2 or 3 on the ring and may be optionally independently substituted with one or more lower alkyl or alkenyl, hydroxy, alkoxy, alkylthio, mercapto, halogen, haloalkyl, amino, monosubstituted amino, or disubstituted amino (substituents as indicated for group R3 in Table 1). 
The term xe2x80x9cpyridylxe2x80x9d refers to a group which has the core ring structure of Structure C. The pyridyl group may be attached to the rest of the molecule through position 2, 3, or 4 on the ring and may be optionally substituted independently with lower alkyl, or alkenyl, hydroxy, alkoxy, alkylthio, mercapto, halogen, haloalkyl, amino, monosubstituted amino, or disubstituted amino (substituents as indicated for group R3 in Table 1). 
The term xe2x80x9cquinolylxe2x80x9d refers to a group having the core ring structure below. The quinolyl group may be attached to the rest of the molecule through positions 2,3,4,5,6,7, or 8. The group may optionally be independently substituted by one or more groups as indicated for the thienyl group above and in Table 1 for the group R3. 
The term xe2x80x9cisoquinolylxe2x80x9d refers to a group with core ring structure shown below. The isoquinolyl group may be attached to the rest of the molecule through positions 1,3,4,5,6,7, or 8. The group may optionally be substituted independently by one or more groups as indicated for the thienyl group above and in Table 1 for the group R3. 
The term xe2x80x9cquinazolylxe2x80x9d refers to a group which has the core ring structure below. 
The group may be attached to the rest of the molecule through positions 1,3,5,6,7, or 8. The group may optionally be substituted independently by one or more groups as indicated for the thienyl group above and in Table 1 for the group R3.
xe2x80x9cMercaptoxe2x80x9d or xe2x80x9cthiolxe2x80x9d refers to the group xe2x80x94SH.
The term xe2x80x9ctetrahydronaphthylxe2x80x9d refers to a group which has a core ring structure of a phenyl ring fused to a cyclohexyl ring, as shown in the structure below, where the attachment to the rest of the molecule can be on either the phenyl ring or on the cyclohexyl ring. The group may optionally be substituted independently as indicated in Table 1 for the group R3. 
The term xe2x80x9cindanylxe2x80x9d refers to a group which has the core bicyclic ring structure below. 
which may optionally be independently substituted as indicated in Table 1 for the group R3. The group may be attached to the rest of the molecule through either the phenyl ring or through the 5-membered ring.
The term xe2x80x9carylalkylxe2x80x9d refers to a lower alkyl group substituted with an aryl group. An example of an arylalkyl group is benzyl where a methyl group is substituted with phenyl. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, allylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The arylalkyl group may be aryl-substituted where the aryl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cthienylalkylxe2x80x9d refers to a lower alkyl group substituted with a thienyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The thienylalkyl group may be thienyl-substituted where the thienyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cfurylalkylxe2x80x9d refers to a lower alkyl group substituted with a furyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The furylalkyl group may be furyl-substituted where the furyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cpyridylalkylxe2x80x9d refers to a lower alkyl group substituted with a pyridyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The pyridylalkyl group may be pyridyl-substituted where the pyridyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzothienylalkylxe2x80x9d refers to a lower alkyl group substituted with a benzothienyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The benzothienylalkyl group may be benzothienyl-substituted where the benzothienyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cindolyalkylxe2x80x9d refers to a lower alkyl group substituted with an indole group. The lower allyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The indolyalkyl group may be indole-substituted where the indole group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cquinolinylalkylxe2x80x9d refers to a lower alkyl group substituted with an quinolinyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The quinolinylalkyl group may be quinolinyl-substituted where the quinolinyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cisoquinolinylalkylxe2x80x9d refers to a lower alkyl group substituted with an isoquinolinyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The isoquinolinylalkyl group may be isoquinolinyl-substituted where the quinolinyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cquinoxalinylalkylxe2x80x9d refers to a lower alkyl group substituted with an quinoxalinyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The quinoxalinylalkyl group may be quinoxalinyl-substituted where the quinolinyl group is optionally substituted with a lower allyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cquinazolinylalkylxe2x80x9d refers to a lower alkyl group substituted with an quinazolinyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The quinazolinylalkyl group may be quinazolinyl-substituted where the quinazolinyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzimidazolylalkylxe2x80x9d refers to a lower alkyl group substituted with an benzimidazolyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The benzimidazolylalkyl group may be benzimidazolyl-substituted where the quinazolinyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzothiazolylalkylxe2x80x9d refers to a lower alkyl group substituted with an benzothiazolyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, allylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The benzothiazolylalkyl group may be benzothiazolyl-substituted where the quinazolinyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzoxazolylalkylxe2x80x9d refers to a lower alkyl group substituted with an benzoxazolyl group. The lower alkyl group may be optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. The benzoxazolylalkyl group may be benzoxazolyl-substituted where the benzoxazolyl group is optionally substituted with a lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzofuranylxe2x80x9d refers to a group which has the core ring structure of Structure A. The benzofuranyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzothienylxe2x80x9d refers to a group which has the core ring structure of Structure B. The benzothienyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cindolylxe2x80x9d refers to a group which has the core ring structure of Structure C. The indolyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzimidazolylxe2x80x9d refers to a group which has the core ring structure of Structure D. The benzimidazolyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzothiazolylxe2x80x9d refers to a group which has the core ring structure of Structure F. The benzothiazolyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino.
The term xe2x80x9cbenzoxazolylxe2x80x9d refers to a group which has the core ring structure of Structure E. The benzoxazolyl group may be optionally substituted with lower alkyl, alkoxy, alkylthio, halogen, amino, monosubstituted amino, or disubstituted amino. 
The term xe2x80x9cxcex1-amino acylxe2x80x9d refers to a group RCH(NR1R2)C(O)xe2x80x94 where NR1R2 is an optionally substituted amino group and R is H or a saturated or unsaturated hydrocarbon, preferably of 1-6, more preferably 1-4 carbon atoms. The term xe2x80x9cxcex2-amino acylxe2x80x9d refers to a group Rxe2x80x94CH(NR1R2)CH2C(O)xe2x80x94, where the components are as just described.
The term xe2x80x9c(alpha-aminoacyl)amidoxe2x80x9d refers to a group having an amide linkage and which is alpha-amino substituted. Preferably the group is an amide-linked alpha-amino acid, which may optionally be substituted, for example, glycylamido, D-alanylamido, D-aspartylamido, D-glutamylamido, D-leucylamido, D-phenylalanylamido, D-phenylglycylamido, D-tyrosylamido.
The term xe2x80x9caminoalkylxe2x80x9d refers to an amino substituted lower alkyl group, preferably (CH2)nRbRc where n=1-4; Rb and/or Rc is H, lower alkyl, aryl.
In preferred embodiments, certain efflux pump inhibitors of the present invention have structures which are shown by the generic structure in Table 2 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration; even racemic materials fulfill the structural generics description.
In preferred embodiments, certain efflux pump inhibitors of the present invention have structures which are shown by the generic structure in Table 3 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration; even racemic materials fulfill the structural generics description.
In preferred embodiments, certain efflux pump inhibitors of the present invention also have structures which are shown by the generic structure in Table 4 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration; even racemic materials fulfill the structural generics description.
In preferred embodiments, certain efflux pump inhibitors of the present invention also have structures which are shown by the generic structure in Table 5 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration; even racemic materials fulfill the structural generics description.
In preferred embodiments, certain efflux pump inhibitors of the present invention also have structures which are shown by the generic structure in Table 6 below:
Where there are centers of asymmetry, the absolute stereochemistry can be either R or S-configuration and any combination of configuration; even racemic materials fulfill the structural generics description.
The generic compound descriptions above should be understood to specifically include additional narrower generic descriptions in which the possible substituents for one or more of the specified substituent groups or substitutions is limited to a subset of the listed groups.
In the claims, the substituents and moieties for the compounds described by sub-generic structures 2, 3, 4, 5, and 6 are as described for Structure 1 unless specified differently.
Compounds within the generic description above can be obtained by synthetic chemistry methods known to those skilled in the chemical arts as exemplified in the Examples below. Specific compound examples within the generic description are provided in the Detailed Description below.
A particularly appropriate example of a microbe appropriate for the use of an efflux pump inhibitor is a pathogenic bacterial species, Pseudomonas aeruginosa, which is intrinsically resistant to many of the commonly used antibacterial agents. Exposing this bacterium to an efflux pump inhibitor can significantly slow the export of an antibacterial agent from the interior of the cell or the export of siderophores. Therefore, if another antibacterial agent is administered in conjunction with the efflux pump inhibitor, the antibacterial agent, which would otherwise be maintained at a very low intracellular concentration by the export process, can accumulate to a concentration which will inhibit the growth of the bacterial cells. This growth inhibition can be due to either bacteriostatic or bactericidal activity, depending on the specific antibacterial agent used. While P. aeruginosa is an example of an appropriate bacterium, other bacterial and microbial species may contain similar broad substrate pumps, which actively export a variety of antimicrobial agents, and thus can also be appropriate targets.
In addition as suggested above, for some microbial, e.g., bacterial, species, efflux pump inhibitors can decrease the virulence of the microbe, for example, by inhibiting the transport of factors important for pathogenicity. Again using P. aeruginosa as an example, inhibition of an efflux pump in this bacterium inhibits the uptake of iron, which is important for pathogenicity. The mechanism of bacterial iron transport involves molecules called siderophores, which are synthesized and exported by bacterial cells via efflux pumps. These siderophores bind tightly to iron scavenged from the host, and are then taken up by the bacteria. In this way, the iron needed for bacterial metabolism is obtained, and an infection can be maintained.
Therefore, illustrating the utility of efflux pump inhibitors, inhibiting the efflux pump of P. aeruginosa allows obtaining one or more of the following biological effects:
1. P. aeruginosa strains will become susceptible to antibiotics that could not be used for treatment of pseudomonad infections, or become more susceptible to antibiotics which do inhibit pseudomonal growth.
2. P. aeruginosa strains will become more susceptible to antibiotics currently used for treatment of pseudomonad infections.
3. Virulence of P. aeruginosa will be attenuated because the availability of iron will be hampered.
4. The inhibition of the pump or of one of the components of the pump may be lethal or prevent growth.
Obtaining even one of these effects provides a potential therapeutic treatment for infections by this bacterium. Also, as previously mentioned, similar pumps are found in other microorganisms. Some or all of the above effects can also be obtained with those microbes, and they are therefore also appropriate targets for detecting or using efflux pump inhibitors. Thus, the term xe2x80x9cmicrobesxe2x80x9d include, for example, bacteria, fuingi, yeasts, and protozoa.
As indicated, the bacterium to be inhibited through the use of an efflux pump inhibitor can be from other bacterial groups or species, such as one of the following: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigellaflexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saccharolyticus. 
The term xe2x80x9cefflux pumpxe2x80x9d refers to a protein assembly which exports substrate molecules from the cytoplasm or periplasm of a cell, in an energy dependent fashion. Thus an efflux pump will typically be located in the cytoplasmic membrane of the cell (spanning the cytoplasmic membrane). In Gram-negative bacteria the pump may span the periplasmic space and there may also be portion of the efflux pump which spans the outer membrane. Certain efflux pumps will include a polypeptide which has at least 50% amino acid sequence similarity with a polypeptide which is part of the Pseudomonas aeruginosa mexA/mexB/oprM efflux pump or the efflux pump overexpressed by P. aeruginosa Strain K385, or the efflux pump overexpressed by P. aeruginosa Strain PAO4098E. Due to the described sequence similarity of a component polypeptide of the efflux pump, such an efflux pump is termed a Pseudomonas aeruginosa-type efflux pump.
The term xe2x80x9cnon-tetracycline-specific efflux pumpxe2x80x9d refers to an efflux pump which is not highly specific for tetracycline (relative to other antibiotics) and thus is not a tetracycline (tetracycline-specific) efflux pump. The term thus includes broad substrate pumps (efflux a number of compounds with varying structural characteristics) and pumps which are highly specific for compounds (including antibiotics) other than tetracyclines. Tetracycline efflux pumps are involved in specific resistance to tetracycline in bacteria. (Speer et al., 1992, Clin. Microbiol. Rev. 5: 387-399.) As noted, these pumps are highly specific for tetracyclines, and their presence confers high tetracycline resistance to the cell. However, they do not confer resistance to other antibiotics. The genes for the tetracycline pump components are found in plasmids in Gram-negative as well as in Gram-positive bacteria and can be divided in two main groups, tetA(A-E), and tetK and tetL. TetA-E tetracycline resistance determinants contain a structural gene, tetA, which is a tetracycline specific pump, and a repressor gene, tetR, that mediates inducible resistance to tetracyclines. Tetracycline efflux pumps belonging to this group are designated tetA(A), teA(B), tetA(D), and tetA(), and are found in Enterobacteriaceae and other Gram-negative bacteria TetK and TetL are pumps involved in tetracycline resistance in Gram-positive bacteria. The genes are regulated via translational attenuation and are not homologous to tetA group.
An xe2x80x9cefflux pump inhibitorxe2x80x9d is a compound which specifically interferes with the ability of an efflux pump to export its normal substrate, or other compounds such as an antibiotic. The inhibitor may have intrinsic antimicrobial (e.g., antibacterial) activity of its own, but at least a significant portion of the relevant activity is due to the efflux pump inhibiting activity. Of particular interest in this invention, are compounds which inhibit the export or activity of efflux pumps which have a broad substrate range which includes antibacterial agents. The term xe2x80x9cnon-tetracycline-specific efflux pump inhibitorxe2x80x9d refers to an efflux pump inhibitor which inhibits a non-tetracycline-specific efflux pump. The term xe2x80x9cPseudomonas aeruginosa-type efflux pump inhibitorxe2x80x9d refers to an efflux pump inhibitor which inhibits a Pseudomonas aeruginosa-type efflux pump. A xe2x80x9cPseudomonas aeruginosa efflux pump inhibitorxe2x80x9d is an efflux pump inhibitor which inhibits the export activity of an efflux pump found in Pseudomonas aeruginosa. 
By xe2x80x9ccomprisingxe2x80x9d is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
In another aspect, this invention provides a method for treating a microbial infection, e.g., a bacterial infection, in an animal by administering to an animal suffering from such an infection an efflux pump inhibitor as described above in an amount sufficient to reduce efflux pump activity.
In a preferred embodiment, the inhibitor is one which decreases the pathogenicity of the microbe. Such a decrease in pathogenicity can be obtained, for example, by interfering with bacterial iron acquisition by inhibiting the transport of siderophores. The pathogenicity may also be reduced by reducing or eliminating the microbial products which cause tissue-damaging effects to the host. Other methods of reducing pathogenicity are, however, also within this aspect. The animal may be, for exanple, chickens and turkeys, and in certain preferred embodiments is a mammal, e.g., a human.
In certain preferred embodiments, the microbial infection may be due to bacteria, which may, for example, be any of the bacterial species indicated above, but specifically including Pseudomonas aeruginosa. 
In a related aspect, this invention provides a method of treating an animal suffering from a microbial infection by administering to the animal an efflux pump inhibitor in an amount sufficient to reduce efflux pump activity. In this aspect, the efflux pump inhibitor in one which reduces the in vivo viability of a microbe involved in the infection. By reducing the in vivo viability, the infected animal can more readily clear its body of the infection, or the microbes may even be killed. In particular embodiments the animal is a mammal. Also in particular embodiments, the microbe may be from one of a variety of pathogenic bacterial species, specifically including those listed above.
The term xe2x80x9cin vivo viabilityxe2x80x9d refers to the ability of a microbe, e.g., a bacterium, to survive or grow in a host, such as an animal. Therefore, an efflux pump inhibitor which reduces the in vivo viability of a microbe may stop the growth of the microbe and/or kill the microbe. Such efflux pump inhibitors, therefore are antimicrobial agents.
In a further related aspect, this invention includes a method for prophylactic treatment of an animal, e.g., a mammal. In this method, an efflux pump inhibitor which reduces the pathogenicity of a microbe is administered to a mammal at risk of a microbial infection, e.g., a bacterial infection.
In a related aspect, the invention provides a method for treating a microbial infection in an animal, specifically including in a mammal, by treating an animal suffering from such an infection with an antimicrobial agent and an efflux pump inhibitor which increase the susceptibility of the microbe for that antimicrobial agent. In this way a microbe involved in the infection can be treated using the antimicrobial agent in smaller quantities, or can be treated with an antimicrobial agent which is not therapeutically effective when used in the absence of the efflux pump inhibitor. Thus, this method of treatment is especially appropriate for the treatment of infections involving microbial strains which are difficult to treat using an antimicrobial agent alone due to a need for high dosage levels (which can cause undesirable side effects), or due to lack of any clinically effective antimicrobial agents. However, it is also appropriate for treating infections involving microbes which are susceptible to particular antimicrobial agents as a way to reduce the dosage of those particular agents. This can reduce the risk of side effects, but can also reduce the selection effect for highly resistant microbes resulting from the consistent high level use of a particular antimicrobial agent. In particular embodiments the microbe is a bacterium, which may, for example, be from any of the groups or species indicated above Also in particular embodiments various antibacterial agents can be used. These include quinolones, tetracyclines, glycopeptides, aminoglycosides, beta-lactams, rifamycins, coumermycins, macrolides, and chloramphenicol. In particular embodiments an antibiotic of the above classes can be, for example, one of the following:
Beta-Lactam Antibiotics
imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefinenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceflibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefmetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763
Macrolides
azithromycin, clarithromycin, erytiromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin
Quinolones
amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, PD131628, PD138312, PD140248, Q-35, AM-1 155,NM394, T-3761, rufloxacin, OPC-17116, DU-6859a (identified in Sato, K. et al., 1992, Antimicrob Agents Chemother. 37:1491-98), DV-7751a (identified in Tanaka, M. et al., 1992, Antimicrob. Agents Chemother. 37:2212-18)
Tetracyclines and Oxazolidinones
chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, lenazolide, eperozolid
Aminoplycosides
amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, clindamycin, lincomycin.
In a further related aspect, this invention includes a method for prophylactic treatment of a mammal. In this method, an antimicrobial agent and an efflux pump inhibitor is administered to a mammal at risk of a microbial infection, e.g., a bacterial infection.
In the context of the response of a microbe, such as a bacterium, to an anti-microbial agent, the term xe2x80x9csusceptibilityxe2x80x9d refers to the sensitivity of the microbe for the presence of the antimicrobial agent. So, to increase the susceptibility means that the microbe will be inhibited by a lower concentration of the antimicrobial agent in the medium surrounding the microbial cells. This is equivalent to saying that the microbe is more sensitive to the antimicrobial agent. In most cases the minimum inhibitory concentration (MIC) of that antimicrobial agent will have been reduced.
As used herein, the term xe2x80x9ctreatingxe2x80x9d refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. The term xe2x80x9cprophylactic treatmentxe2x80x9d refers to treating a patient who is not yet infected, but who is susceptible to, or otherwise at risk of, a particular infection. The term xe2x80x9ctherapeutic treatmentxe2x80x9d refers to administering treatment to a patient already suffering from an infection. Thus, in preferred embodiments, treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of a potentiator and an antibacterial (or antimicrobial) agent in combination (either simultaneously or serially).
By xe2x80x9ctherapeutically effective amountxe2x80x9d or xe2x80x9cpharmaceutically effective amountxe2x80x9d is meant an amount of an efflux pump inhibitor, or amounts individually of an efflux pump inhibitor and an antimicrobial agent, as disclosed for this invention, which have a therapeutic effect, which generally refers to the inhibition to some extent of the normal metabolism of microbial cells causing or contributing to a microbial infection. The doses of efflux pump inhibitor and antimicrobial agent which are useful in combination as a treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of efflux pump inhibitor and antimicrobial agent which, when used in combination, produce the desired therapeutic effect as judged by clinical trial results and/or model animal infection studies. In particular embodiments, the efflux pump inhibitor and antimicrobial agent are combined in pre-determined proportions and thus a therapeutically effective amount would be an amount of the combination. This amount and the amount of the efflux pump inhibitor and antimicrobial agent individually can be routinely determined by one of skill in the art, and will vary, depending on several factors, such as the particular microbial strain involved and the particular efflux pump inhibitor and antimicrobial agent used. This amount can further depend upon the patient""s height, weight, sex, age and medical history. For prophylactic treatments, a therapeutically effective amount is that amount which would be effective if a microbial infection existed.
A therapeutic effect relieves, to some extent, one or more of the symptoms of the infection, and includes curing an infection. xe2x80x9cCuringxe2x80x9d means that the symptoms of active infection are eliminated, including the elimination of excessive members of viable microbe of those involved in the infection. However, certain long-term or permanent effects of the infection may exist even after a cure is obtained (such as extensive tissue damage).
The term xe2x80x9cmicrobial infectionxe2x80x9d refers to the invasion of the host mammal by pathogenic microbes. This includes the excessive growth of microbes which are normally present in or on the body of a mammal. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a mammal is xe2x80x9csufferingxe2x80x9d from a microbial infection when excessive numbers of a microbial population are present in or on a mammal""s body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal. Specifically, this description applies to a bacterial infection.
The term xe2x80x9cadmninistrationxe2x80x9d or xe2x80x9cadministeringxe2x80x9d refers to a method of giving a dosage of an antimicrobial pharmaceutical composition to a mammal, where the method is, e.g., topical, oral, intravenous, intraperitoneal, or intramuscular. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the microbe involved, and the severity of an actual microbial infection.
The term xe2x80x9cmammalxe2x80x9d is used in its usual biological sense. Thus, it specifically includes humans, cattle, horses, dogs, and cats, but also includes many other species.
In another aspect, this invention also features a method of inhibiting a membrane channel in a cellular membrane, involving contacting the membrane channel with a membrane channel inhibitor, where the inhibitor reduces the effluxing capacity of the membrane channel. In specific embodiments, at least one polypeptide of the membrane channel has at least 50% amino acid sequence similarity with a polypeptide of the mexA/mexB/oprM efflux pump, or of the efflux pump overexpressed by Pseudomonas aeruginosa Strain K385.
As used herein, the term xe2x80x9cmembrane channelxe2x80x9d refers to a protein assembly located in the cellular membrane of a cell which allows the transport of one or more types of molecules across the membrane. Such transport may be either passive transport in response to concentration gradients, or may be active transport which depends upon a cellular energy source.
A xe2x80x9cmembrane channel inhibitorxe2x80x9d then is, similar to an efflux pump inhibitor, a compound which slows or prevents the transport of molecules across the cellular membrane using the corresponding membrane channel.
This invention also features a method of enhancing the antimicrobial activity of an antimicrobial agent against a microbe, in which such a microbe is contacted with an efflux pump inhibitor, e.g., a non-tetracycline specific efflux pump inhibitor, to an efflux pump in the cell, and an antibacterial agent. The efflux pump inhibitor is a compound as described above. Thus, this method makes an antimicrobial agent more effective against a cell which expresses an efflux pump when the cell is treated with the combination of an antimicrobial agent and a non-tetracycline-specific efflux pump inhibitor. In particular embodiments the microbe is a bacterium or a fungus, such as any of those indicated in the first aspect above; the antibacterial agent can be selected from a number of structural classes of antibiotics including, e.g., beta-lactams, glycopeptides, aminoglycosides, quinolones, tetracyclines, rifamycins, coumermycins, macrolides, and chloramphenicol. In particular embodiments an antibiotic of the above classes can be as stated above.
In a further aspect this invention provides pharmaceutical compositions effective for treatment of an infection of an animal, e.g., a mammal, by a microbe, such as a bacterium or a fungus. The composition includes a pharmaceutically acceptable carrier and an efflux pump inhibitor as described above. In preferred embodiments, such compositions contain efflux pump inhibitors which are themselves effective antimicrobial agents, even in the absence of another antimicrobial agent (i.e., have intrinsic antimicrobial activity). Thus, pharmaceutical compositions including such efflux pump inhibitors can be used either alone or in conjunction with another antimicrobial agent. Also in preferred embodiments, the efflux pump inhibitors in pharmaceutical compositions of this aspect are efflux pump inhibitors which enhance the effectiveness of an antimicrobial agent other than the efflux pump inhibitor, so such compositions would generally be used in combination with such other antimicrobial agent. The invention also provides pharmaceutical compositions similarly effective for treatment of an infection of a mammal which include an efflux pump inhibitor and an antimicrobial agent. Similarly, the invention provides antimicrobial formulations which include an antimicrobial agent, an efflux pump inhibitor, and a carrier. In preferred embodiments, the antimicrobial agent is an antibacterial agent.
A xe2x80x9ccarrierxe2x80x9d or xe2x80x9cexcipientxe2x80x9d is a compound or material used to facilitate administration of the compound, for example, to increase the solubility of the compound. Solid carriers include, e.g., starch, lactose, dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g., sterile water, saline, buffers, non-ionic surfactants, and edible oils such as oil, peanut and sesame oils. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck and Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman""s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.
In yet another aspect, the invention provides a method of suppressing growth of a microbe, e.g., a bacterium, expressing an efflux pump, e.g., a non-tetracycline-specific efflux pump. As illustrated by the case where the microbe is a bacterium, the method involves contacting that bacterium with an efflux pump inhibitor, e.g., a non-tetracycline-specific efflux pump inhibitor, in the presence of a concentration of antibacterial agent below the MIC of the bacterium. This method is useful, for example, to prevent or cure contamination of a cell culture by a bacterium possessing an efflux pump. However, it applies to any situation where such growth suppression is desirable.
In a related aspect, the invention provides a method of suppressing growth of a microbe, e.g., a bacterium, which involves contacting the microbe with an efflux pump inhibitor which reduces the expression of a component of an efflux pump. Such an inhibitor can act on the regulation of that expression in number of different ways. It may, for example, enhance the production of a repressor molecule which prevents expression of an efflux pump component. Another possible mechanism is if the inhibitor blocks the release of a repressor molecule. Examples of such a repressor is MarR in E. coli (Seoane and Levy, 1994, Abstr. of the Am. Soc. for Microbiol. Gen. Meeting, Las Vegas, Nev., Abstr. H-26). An example of a positive regulator is BmrR in Bacillus subtilis (Ahmed et al., 1994, J. Biol. Chem.).
In another related aspect, the invention provides a method for reducing a population of a microbe, e.g., a bacterial strain, involving contacting the population with an efflux pump inhibitor which inhibits a component of an efflux pump expressed in the microbe in that population, which is essential for the growth of the microbe expressing that efflux pump. In particular embodiments, that component is a cytoplasmic membrane component. As indicated above, such efflux pump inhibitors may act in various ways, including, but not limited to, acting directly on the essential component, or acting to inhibit the expression of that component.
The term xe2x80x9creducing a populationxe2x80x9d means that the microbes of that population are being killed. This is distinguished from the action of a static agent, e.g., a bacteriostatic agent, which prevents the bacteria from growing and multiplying but does not kill the microbes. Accordingly, in the context of this aspect, an xe2x80x9cessential componentxe2x80x9d of an efflux pump is one which is essential to the in vivo survival of the microbe, i.e., the survival in a host.
In yet another aspect, this invention provides a method for enhancing growth of an animal by administering an efflux pump inhibitor to the animal, which inhibits an efflux pump expressed in a bacterial strain in the animal, and which inhibits the growth of that bacterial strain. Such a growth enhancing effect may result from the reduced energy consumption by the bacteria, which increases the food energy available to the animal. This method is appropriate, for example, for use with cattle, swine, and fowl such as chickens and turkeys.
In an additional aspect, the invention provides novel compounds having efflux pump activity. These compounds have chemical structures as described above.
As indicated above, while the present invention is presently exemplified by activity against bacteria, compounds of the present invention also have activity against other microbes, for example against yeasts and/or other fungi. Thus, the above aspects also include embodiments in which described compounds are active or effective against such other microbes.
In a further aspect, the invention provides a method of making a pharmaceutical composition comprising the steps of identifying an efflux pump inhibitor having a chemical structure as set forth in Structure 1 or Structure 2, or the sub-generic structures 3, 4, 5, and 6, as described herein; synthesizing said efflux pump inhibitor in an amount sufficient to provide a therapeutic response; and preparing a pharmaceutical composition containing said efflux pump inhibitor. The efflux pump inhibitor may have the chemical structure as described above. The pharmaceutical composition may also contain one or more antimicrobial agents, e.g., as identified above, and one or more carriers, diluents, and excipients. Further, in preferred embodiments, the efflux inhibitor compound is active against a microbe, e.g., a bacteriam, as identified above.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
Identification of Efflux Pump Inhibitors
Initial identification of efflux pump inhibitors having structures as described was performed using a screening method as generally described in Trias et al., EFFLUX PUMP INHIBITORS, U.S. application Ser. No. 08/427,088; Trias et al., EFFLUX PUMP INHIBITORS, U.S. application No. not yet assigned, filed Jul. 22, 1997; and Trias et al., International Publication WO96/05469. In particular, the screening method based on inhibition of microbial growth in the presence of a subinhibitory concentration of an antibacterial agent which is normally effluxed by the test microbe and a concentration of a test compound was used for identifying some of the active compounds disclosed herein. In this method, inhibition of growth of the microbe is indicative that export of the antibacterial agent is inhibited by the test compound, and that the test compound is therefore an efflux pump inhibitor. The mode of action of the test compound so identified can then be confirmed as inhibiting active efflux. However, other screening methods for detecting efflux pump inhibitors can also be used, specifically including the additional methods described in the above references.
Synthesis of Derivatives of Efflux Pump Inhibitors from Screening
Exemplary compounds of the present invention were synthesized by methods as described in the Examples below. Those skilled in the art will understand how to synthesize additional compounds within the scope of this invention based on the described syntheses and the knowledge of those skilled in the art of chemical synthesis.
Susceptibility Testing
Particular exemplary efflux pump inhibitor compounds within the generic descriptions of the compounds of this invention were evaluated for potentiation effect. The in vitro microbiological data for antibiotic potentiation is presented in Tables 7 and 8 below.
For compounds labeled with the prefix xe2x80x98Mxe2x80x99, potentiation effect is observed by the reduction of the minimum inhibitory concentration of levofloxacin in the presence of the experimental efflux pump inhibitor. The activity of efflux pump inhibitors (EPI) in combination with fluoroquinolones, such as levofloxacin, is assessed by the checkerboard assay (Antimicrobial Combinations. In Antibiotics in Laboratory Medicine, Ed. Victor Lorian, M.D., Fourth edition, 1996, pp 333-338) using broth microdilution method performed as recommended by the National Committee for Clinical Laboratory Standards (1997). Methods for Dilution of Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobicallyxe2x80x94Fourth Edition; Approved Standard. NCCLS Document M7-A4, Vol 17 No.2). The test organism used is Pseudomonas aeruginosa PAM1001. The compounds of this invention demonstrate pump inhibitory activity against a broad-range of P. aeruginosa over-producing singular efflux pumps (MexAB, MexCD, and MexEF) and clinical strains containing multiple efflux pumps, not limited to the Mex classification. The compounds tabulated below are representative of the described invention.
In these assays, multiple dilutions of two drugs, namely the EPI and levofloxacin, are being tested, alone and in combination, at concentrations equal to, above and below their respective minimal inhibitory concentrations (MICs). In the case of EPI, most of these compounds are devoid of intrinsic antimicrobial activity and are tested up to the maximum concentration of 40 xcexcg/mL. The MIC of levofloxacin against P. aeruginosa PAM1001 is 4 xcexcg/mL.
The EPI tested are readily soluble in water and stock solutions are prepared at a final concentration of 2 mg/mL. Stock solutions are further diluted, according to the needs of a particular assay, in Mueller Hinton Broth (MHB). Stock solution can be stored at xe2x88x9280xc2x0 C. Quinolones are solubilized according to the instructions of the manufacturers, at a concentration of 1 mg/mL. They are then further diluted in MHB. Stock solution can be stored at xe2x88x9280xc2x0 C.
The checkerboard assay is performed in microtiter plates. Levofloxacin is diluted in the x axis, each column containing a single concentration of levofloxacin. The EPI is diluted in the y axis, each row containing an equal concentration of EPI. The result of these manipulations is that each well of the microtiter plate contains a unique combination of concentrations of the two agents. Each EPI are tested independently.
The assay is performed in MHB with a final bacterial inoculum of 5xc3x97105 CFU/mL (from an early-log phase culture). Microtiter plates are incubated during 20 h at 35xc2x0 C. and are read using a microtiter plate reader (Molecular Devices) at 650 nm as well as visual observation using a microtiterplate reading mirror. The MIC is defined as the lowest concentration of quinolone, within the combination, at which the visible growth of the organism is completely inhibited.
For compounds labeled with the prefix xe2x80x98Dxe2x80x99, potentiation is measured by inhibition of growth of Pseudomonas aeruginosa PAM1001 at varying concentrations of efflux pump inhibitors (EPIs) in the presence of levofloxacin (0.25 microg/mL). Bacteria are grown in Mueller Hinton Broth (MHB, Difco) with an initial inoculum of 1xc3x97106 CFU/mL. Kinetics of bacterial growth is monitored, during 18 h of incubation at 37xc2x0 C., by measurement of the culture absorbance at 650 nm using a 96-well plate spectrophotometer (SpectraMax, Molecular Devices). The inhibitory effect of EPIs is expressed as a percentage of growth inhibition compared to a control bacterial culture grown in the presence of 0.25 microg/mL levofloxacin but without EPIs.
In vivo Evaluation of Efflux Pump Inhibitor Compounds
Inhibitors of the bacterial efflux pumps are generally initially characterized in vitro. Those which show effective inhibition of the pump(s) and which show synergistic activity with antibiotics are selected for evaluation in vivo. Efficacy testing will be done using standard procedures. Primary efficacy evaluation may be done using the murine septicemia model (M. G. Bergeron, 1978, Scand. J. Infect. Dis. Suppl. 14:189-206; S. D. Davis, 1975, Antimicrob. Agents Chemother. 8:50-53). In this model a supra-lethal dose of bacteria is used to challenge the rodents. Treatment is initiated, varying either or both time(s) of treatment and dose of antibiotic. In these experiments both the antibiotic and the efflux pump inhibitor doses are varied. A positive result is indicated by significant increase in protection from the lethal infection by the combination of the potentiator (the efflux pump inhibitor) and the antibiotic versus the antibiotic alone.
A second efficacy model which is used is the mouse soft tissue infection model (Vogelman et al., 1988, J. Infect. Dis. 157:287-298). In this model anesthetized mice are infected with an appropriate titer of bacteria in the muscle of the hind thigh. Mice are either neutropenic (cyclophosphamide treated at 125 mg/kg on days xe2x88x924, xe2x88x922, and 0) or immunocompetent. The infecting dose is commonly 105-106 colony forming units per animal. Treatment with the combination of the efflux pump inhibitor and/or antibiotics follows infection, or can occur before infection. The proliferation (or death) of the bacteria within the thigh muscle is monitored over time. Effective combinations show greater activity than the antibiotic alone. Activity is defined as reduction in growth rate of the test bacteria in the murine tissue.
Another model useful for assessing the effectiveness of the efflux pump inhibitors is the diffusion chamber model (Malouin et al., 1990, Infect. Immun. 58:1247-1253; Day et al., J. Infect. 2:39-51; Kelly et al., 1989, Infect. Immun. 57:344-350). In this model rodents have a difflusion chamber surgically placed in their peritoneal cavity. The chamber can consist of a polypropylene cylinder with semipermeable membranes covering the cylinder ends. Difflusion of peritoneal fluid into and out of the chamber provides nutrients for the microbes. The proliferation of the bacteria in the presence and absence of the antibiotic/efflux pump inhibitor is compared to the antibiotic alone. Dose ranging of the combination and the antibiotic alone are done to assess effectiveness of the antimicrobial and combinations.
A tertiary model useful as a stringent test of the efflux pump inhibitor/antibiotic combination is the endocarditis model (J. Santoro and M. E. Levinson, 1978, Infect. Immun. 19:915-918). Either rats or rabbits are effectively used in this model. The effectiveness of combinations of efflux inhibitor and antibiotic are compared to antibiotic alone. The end point is usually viable cells remaining in the cardiac vegetations at the end of treatment.
The examples of infection models provided are not limiting. As understood by those skilled in the art, other models can be utilized as appropriate for a specific infecting microbe. In particular, cell-based infection models may be used in some circumstances instead of animal models.
Pharmaceutical Compositions and Modes of Administration
The particular compound that is an efflux pump inhibitor can be administered to a patient either by itself, or in combination with an antimicrobial, e.g., antibacterial, agent, or in pharmaceutical compositions where it is mixed with a suitable carrier(s) or excipient(s) or diluent(s). A combination of an efflux pump inhibitor with an antimicrobial agent can be of at least two different types. In one, a quantity of an efflux pump inhibitor is combined with a quantity of an antimicrobial agent in a mixture, e.g., in a solution or powder mixture. In such mixtures, the relative quantities of the inhibitor and the antimicrobial agent may be varied as appropriate for the specific combination and expected treatment. In a second type of combination an inhibitor and an antimicrobial agent can be covalently linked in such manner that the linked molecule can be cleaved within the cell. However, the term xe2x80x9cin combinationxe2x80x9d can also refer to other possibilities, including serial administration of an inhibitor and another antimicrobial agent. In addition, an efflux pump inhibitor and/or another antimicrobial agent may be administered in pro-drug forms, i.e. the compound is administered in a form which is modified within the cell to produce the functional form. In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound(s) that results in amelioration of symptoms or a prolongation of survival in a patient, and may include elimination of a microbial infection.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. It is preferable that the therapeutic serum concentration of an efflux pump inhibitor should be in the range of 0.1-100 xcexcg/mL.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.
In particular preferred embodiments, the efflux inhibitor in a pharmaceutical composition has a structure as shown by the generic structures described above.
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition. (See e.g. Fingl et al., in THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 1975, Ch. 1 p. 1). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
Depending on the specific infection being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington""s Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intra-thecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank""s solution, Ringer""s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art, into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.